VLP-based model for the study of airborne viral pathogens

ABSTRACT The recent COVID-19 pandemic has underscored the danger of airborne viral pathogens. The lack of model systems to study airborne pathogens limits the understanding of airborne pathogen distribution as well as potential surveillance and mitigation strategies. In this work, we develop a novel model system to study airborne pathogens using virus-like particles (VLPs). Specifically, we demonstrate the ability to aerosolize VLP and detect and quantify aerosolized VLP RNA by reverse transcription-loop-mediated isothermal amplification in real-time fluorescent and colorimetric assays. Importantly, the VLP model presents many advantages for the study of airborne viral pathogens: (i) similarity in size and surface components; (ii) ease of generation and noninfectious nature enabling the study of biosafety level 3 and biosafety level 4 viruses; (iii) facile characterization of aerosolization parameters; (iv) ability to adapt the system to other viral envelope proteins, including those of newly discovered pathogens and mutant variants; and (v) the ability to introduce viral sequences to develop nucleic acid amplification assays. IMPORTANCE The study and detection of airborne pathogens are hampered by the lack of appropriate model systems. In this work, we demonstrate that noninfectious virus-like particles (VLPs) represent attractive models to study airborne viral pathogens. Specifically, VLPs are readily prepared, are similar in size and composition to infectious viruses, and are amenable to highly sensitive nucleic acid amplification techniques.

more than 250,000 deaths per year worldwide (7,9).Moreover, the recent outbreaks of avian strains harboring HA types H5 or H7, which exhibit mortality rates greater than 40% (i.e., greater than 10× that of the 1918 strain) (10), are particularly concern ing.Fortunately, avian influenza strains have not readily adapted for human-to-human transmission, and thus to date, human fatalities have been relatively rare; however, adaptation of avian strains for human transmission occurs naturally (11).Taken together, coronaviruses and influenza viruses, as well as other respiratory viruses, pose pandemic risks with potentially high mortality rates (5).Consequently, airborne viruses clearly warrant continued efforts for surveillance, mitigation strategies, and the development of efficacious therapeutics and vaccines.

Limitations of current models to study airborne viral pathogens
Bioaerosols have been intensely studied in the laboratory (12,13).Due to biosaf ety concerns, many airborne viral pathogens present challenges for laboratory-based studies.For example, SARS-CoV-2 and avian influenza strains are studied under biosafety level 3 (BSL3) conditions.An alternative approach for the study of viral pathogens under BSL1 and BSL2 conditions, which are standard to most laboratories, would be viruses inactivated by heat, ultraviolet light, or gamma irradiation [e.g., the studies of aerosolized SARS-CoV-2 in reference (14)].However, BSL3 facilities are often required to generate the inactivated viruses, and thus samples are limited in quantity and availability.In addition, treatment by heat denatures proteins present, and thus the surface properties of the virus may be negatively affected, and detection by antibodies may be compromised.Alternatively, ultraviolet light and gamma irradiation treatments result in damage to the viral RNA/DNA, which may preclude detection by nucleic acid amplification techniques or subsequent sequencing efforts.On the other hand, bacteriophages such as MS2 and PR772 are popular models of airborne pathogens due to their biosafe nature, facile preparation, ease of aerosolization due to their relatively small size (20-200 nm in diameter), and detection by standard nucleic acid amplification techniques or cell culture (15).However, there are a number of limitations to the use of aerosolized bacteriophages.First, the size of the phage is often significantly smaller than the virus of interest (e.g., MS2 phage diameter is ~25 nm and SARS-CoV-2 diameter is ~120 nm).Furthermore, many of the popular phages have a very different surface composition, which may be expected to affect aerosolization.For example, phages MS2 and PR772 are unenveloped and thus lack the lipid membrane present in enveloped viruses such as influenza and coronavirus.Accordingly, we propose to develop an alternative model system with attractive biosafety and biological profiles that could be used in studies of airborne viral pathogens.In what follows, we describe our efforts to exploit virus-like particle (VLP) as models for the study of airborne viral pathogens, with a first focus on SARS-CoV-2.

VLP generation and characterization
Plasmids containing SARS-CoV-2 spike and the packaging construct pNL4-3.Luc.R-Ewere co-transfected into 293T cells, as previously described by our laboratories (16)(17)(18).VLPs were quantified by the lateral flow assay (LFA) for HIV p24 (GoStix Plus, Takara).In this case, the number of virions per volume is calculated by the approximate stoichiom etry of p24 in VLP (~2,000/virion).The presence of SARS-CoV-2 spike was verified by a Western blot analysis using the monoclonal antibody CR-3022 (BEI Resources) and comparison to heat-inactivated and gamma-irradiated SARS-CoV-2 (BEI Resources), with equivalent amounts of total protein used.

Aerosolization experiments
The aerosolization experiments were performed with a custom-built cabinet in a biosafety cabinet located in a BSL2 laboratory.Aerosolization of VLP utilized the Bio-Aerosol Nebulizing Generator (BANG) from CH Technologies in the Multi Pass Atomization (MPA) mode.The pressurized airflow into the nebulizer was 4.72 L/min, and the vacuum pump flow rate for collection on the filter was 225 mL/min.Based on p24 concentration, the VLP solution concentration was estimated to be 3.2 × 10 9 virions/mL in PBS buffer.Aerosolized droplets were collected using Millipore 13 mm 0.45 µm MCE filters.Bioaerosol concentration, which is a combination of water droplets and VLPs, was estimated to be 1.33 mg/m 3 using the TSI DustTrak II optical particle counter.The VLPs deposited on the filter were quantified from successive dilutions in the RT-LAMP reaction using a limit of detection (LOD) of 5-30 copies of viral RNA (0.25-1.5 gene copies/µL of assay mixture), based on the plasmid and VLP experiments containing known amounts of DNA or RNA.

Rationale for the VLP model
Virus-like particles, which lack the genetic material for productive infection and are typically derived from the co-transformation of plasmids containing packaging, reporter genes, and/or surface proteins, have been used as surrogates for studying infectious viruses for many years (22).VLPs present numerous advantages with respect to infectious viruses including: (i) relatively safe biosafety profiles; (ii) ease of generation; (iii) similarity in size and surface composition; and (iv) the ability to protect and deliver RNA.Previous uses of VLPs include detailed studies of viral entry mechanisms (16), the use of VLP to discover and characterize novel therapeutics (17,18), use as vaccine vectors, and drug or nucleic acid delivery systems (15,23).

Generation of VLP bearing SARS-CoV-2 spike
As a first step, we generated VLP using the HIV vector pNL43R-E-as the packaging vector and an expression plasmid containing SARS-CoV-2 spike.The packaging vector contains the entire HIV genome with two mutations that result in nonfunctional envelope and rec proteins, and thus the VLPs are considered noninfectious and produced under BSL2 conditions that are standard to many laboratories.One advantage of this particular packaging vector is that the resulting VLPs contain a copy of the viral RNA of the HIV genome, which we will exploit as a marker to track aerosolized viruses by nucleic acid amplification.The addition of the second plasmid containing the SARS-CoV-2 spike in the transfection results in VLP containing an authentic version of the viral envelope protein in the VLP membrane.Consequently, the VLP spikes are almost identical in size [~100 nm (24)] and surface composition (spike and membrane lipids) to infectious SARS-CoV-2.As shown in Fig. 1, Western blot analysis of VLP, heat-inactivated SARS-CoV-2, and gammairradiated SARS-CoV-2 shows the presence of spike in all three samples (we attribute the lower levels of spike observed in the heat-inactivated sample to be due to the use of a conformation-dependent monoclonal antibody).

Development of the LAMP assay using plasmid DNA
In the second step, we developed a nucleic acid amplification assay to track aerosolized VLP.Previously, Curtis et al. ( 21) developed a LAMP-based diagnostic test for HIV using a set of six primers to the HIV pol gene.We first tested the ability of the synthesized primer set to detect the HIV pol gene contained in the packaging vector (cf.Materials and Methods) using the pNL43R-E-plasmid.As shown in Fig. 2a, the real-time LAMP assay shows fluorescence increases in a time-and concentration-dependent manner.Moreover, the concentration (copy number) dependence of the threshold time (Cq) exhibits the expected logarithmic relationship (Fig. 2b), supporting the ability to quantify the amount of nucleic acid present.Interestingly, the slope of the relationship suggests an efficiency of 280% [efficiency = 10 (-1/slope) −1].Notably, efficiencies higher than 100% are often observed in LAMP assays (25)(26)(27).In the next step, we tested the ability to use a colorimetric LAMP assay, which is based on the sensitivity of the dye phenol red to pH changes resulting from nucleic acid amplification (20).As shown in Fig. 3a, the colori metric assay shows the expected transition from pink to yellow over the temperature range of 62°C-68°C.Moreover, using successive dilutions of the template (pNL43R-E-) we estimate that the LOD is less than 12.5 gene copies (0.6 gene copies/µL of assay mixture) for the colorimetric assay (Fig. 3b) and less than 6.75 gene copies (0.3 copies/µL of assay mixture) for the fluorescence assay (Fig. 3c).Note that these experiments suggest that the fluorescence assay is slightly more sensitive than the colorimetric assay at the endpoint; however, the colorimetric assay is advantageous in that it does not require any specialized equipment.

Detection of viral RNA in VLP
As noted above, the VLP used in this study contains ~1 copy of the HIV genome in the form of RNA, which includes the HIV pol gene.Consequently, in the next step, we performed an RT-LAMP assay to detect the viral RNA present in VLP.First, we tested the necessity for pre-treatment of the VLP to liberate the viral RNA.As shown by the real-time RT-LAMP assay in Fig. 4, pre-treatment of the VLP with heat or detergent + heat does not significantly change the sensitivity (i.e., the amount of viral RNA detected).Specifically, the Cq for untreated, heat-treated, or detergent + heat-treated VLP was 11.5 ± 0.4, 10.4 ± 0.2, and 10.4 ± 0.2 min, respectively.In the next step, we assessed the sensitivity of the assay using a series of VLP dilutions of stock concentrations quantified by the ELISA assay for p24.As shown by the colorimetric RT-LAMP assay and the SYBR Green-stained agarose gel of the RT-LAMP reaction, we estimate the LOD to be less than 30 total gene copies of RNA (1.5 gene copies/µL of assay mixture) (Fig. 5a and b).

Aerosolization and detection of VLP viral RNA
In the final step, we designed a biosafety cabinet to study aerosolized VLP.As shown in Fig. 6, the principal components of the cabinet consist of a pressurized air inlet, a Collison nebulizer, and a filter to collect aerosolized VLP.Placing the biosafety cabinet in a biosafety hood located in our BSL2 laboratory, we then aerosolized VLP and collected the aerosolized VLP on an MCE filter (0.45 µm).Subsequently, an extract of the filter was prepared and analyzed for the presence of VLP RNA by RT-LAMP using the assay described above.As shown in Fig. 7a, the colorimetric RT-LAMP assay suggests the presence of the HIV pol gene contained in the VLP RNA in samples 1 and 2, which correspond to undiluted extract and a 1:10 extract dilution.Further analysis by SYBR Green-stained agarose gel confirms the presence of amplified nucleic acid in samples 1 and 2 (Fig. 7b).Consequently, we have shown the ability to collect aerosolized VLP containing SARS-CoV-2 and detect the RNA contained in the VLP by RT-LAMP.

Conclusions
In summary, we have demonstrated the potential of using VLP for the study of airborne viral pathogens.As noted in Introduction, VLP presents a number of attractive properties, including their biosafety profile, facile generation, close physical resemblance to the viral pathogen of interest (SARS-CoV-2 in the present case), and readout assays by nucleic acid amplification techniques, as well as antigen-based techniques (p24 or SARS-CoV-2 spike in the present case).Moreover, different packaging vectors may be used to generate VLP to better mimic the virus of interest (e.g., that of influenza).Alternatively, the sequences for RNA genes under study may be easily cloned into the packaging vector, thereby allowing for detection by various nucleic acid amplification protocols (e.g., in the development of devices to detect airborne viral pathogens).We suggest that this system is ideally suited for detailed studies to characterize the effects of humidity, delivery (e.g., respiration versus coughing models), suspension media (simple buffers versus artificial saliva), and membrane composition (e.g., SARS-CoV-2 spike wild type or variant or the envelope protein from another virus such as influenza hemagglutinin).We note that our groups have recently exploited the VLP model system for the development of a real-time detector of airborne viruses (28).Taken together, we believe that the VLP-based model system will lead to new insights into the aerosolization and infection routes of numerous airborne pathogens, as well as aid in the development of devices to detect airborne viruses.

FIG 1
FIG 1 Western blot analysis for the presence of SARS-CoV-2 spike in VLP and inactivated SARS-CoV-2.For this experiment, monoclonal antibody CR-3022 (BEI Resources) was used with equivalent amounts of total protein in each lane.Heat-inactivated and gamma-irradiated SARS-CoV-2 were obtained from BEI Resources.

FIG 2 FIG 3
FIG 2 Real-time quantitative fluorescent LAMP analysis to detect plasmid.(a) Amplification plots of solutions containing 3.1 × 10 9 , 3.1 × 10 7 , 3.1 × 10 6 , 3.1 × 10 5 , 3.1 × 10 4 , and 3.1 × 10 3 gene copies in 20 µL of assay mixture (left to right).The control experiment, which contains no template, is shown as closed circles.(b) Standard curves for the Cq values versus gene copy number in the reaction mix.Plasmid pNL4-3.Luc.R-E-was used for these experiments.The curves shown represent averages of triplicate experiments, and the error bars represent the estimated standard deviation.

FIG 4
FIG 4 Real-time quantitative fluorescent reverse-transcription LAMP.For these experiments, heat treatment corresponded to the incubation of VLP at 95°C for 10 min before analysis, and detergent treatment corresponded to VLP dilution into a buffer containing 12.5 mM TCEP, 5 mM EDTA, and 0.002% Triton X-100 at pH 8.0.The curves shown represent averages of triplicate experiments.